In affinity assays, a known quantity of a labeled probe competes with or binds to an unknown quantity of unlabeled analyte at binding sites on a target molecule for which the analyte has an affinity. The labeled probe that is bound to the target molecule presents a different measurable phenomenon than the labeled probe that is unbound. Calibration curves relate the presence or quantity of the analyte to the relative amount of bound to unbound labeled probe. The calibration curves are generated by measuring the relative amounts of bound and unbound labeled probe in the presence of known quantities of analyte. In sandwich binding assays, the probe binds to the analyte that is bound to the target molecule. In immunoassays, the analyte is an antigen and the target molecule is an antibody.
Cytokines are important antigens that are secreted by cells to promote a function of the cell. Cytokines control many biological processes including inflammation and disease; therefore, cytokine measurements are widely used in basic research and diagnostics. Cytokines are small regulatory proteins and peptides (with mass in a range from about 8 to about 30 kiloDaltons, kDa, 1 kDa=103 daltons, 1 dalton=one twelfth of the mass of an unbound neutral atom of carbon-12) that exhibit a wide range of biological activities. Cytokines are released in unique profiles in response to inflammation, infection, systemic infections such as sepsis, chronic wound healing, and even as predictors of mortality.
Affinity assays for cytokines secreted by a cell are especially important but are beset by several challenges. Physiological levels are often low; less than 10 picograms per milliliter (pg/ml, where 1 pg=10−12 grams and 1 ml=10−3 liters), which corresponds to a range from about 0.5 picoMolar (pM, 1 pM=10−12 Molar, 1 Molar=1 mole per liter) to about 5 pM. The detection of cytokines is also complicated by spatial heterogeneity of their secretion, rapid turnover and short life-time. Many techniques involve the removal, degradation or death of cells that secrete the cytokines, which make it difficult to assess the rate of secretion or the phenotype associated with a cytokine profile.
A majority of cytokine assays developed over the past decade use specific anti-cytokine antibodies. Currently there are two dominant technologies for the measurement of multiple cytokines in biological samples including cell supernatants: multiplex sandwich ELISA and bead-based assays.
One important aspect of biological and clinical studies is finding correlation of cell phenotypes with the profile of cytokines secreted by these cells. Technologies for single cell assay include flow cytometry or intracellular cytokine cytometry (ICC) and ELISPOT.
Flow cytometry is currently mostly used for measurements of cell surface molecules and intracellular levels of cytokines. There are several advantages of flow cytometry: single cell measurement; multiple biomarker detection; and sorting of cell populations for subsequent analysis. There are also several limitations of cytometry: requirement for expensive equipment and trained personnel; inability to measure secreted proteins from live cells; and difficulties in performing analysis with samples of limited cell number. For cytometry, the cell must be permeabilized and treated with secretion inhibitors which, at the very least, interfere with normal cell function.
ELISPOT assays permit the ex vivo identification of cells actively secreting cytokines and can detect a single cell out of a million, based on well-defined spots that clearly represent numbers of cytokine-secreting cells. Several limitations of ELISPOT assays include: no quantitative information on the level of secreted cytokines; difficult multiplexing (even double cytokines is difficult); and, a multi-step protocol that makes ELISPOT usage difficult. To address the need for double cytokine profiling, a fluorometric modification of ELISPOT (FLUOROSPOT) has been reported. However, because of insufficient fluorescence signal, a complex biochemical amplification is needed which is not convenient for practical use. Simultaneous correlation of cell phenotypes (or cell viability) with cytokine secretion is impossible to realize with ELISPOT or standard fluorometric approaches because of the required washing steps that remove the cells before the imaging process.